Severe Acute Respiratory Syndrome (SARS) has recently emerged as a highly infectious and deadly disease and is caused by a virus that appears to have crossed the species barrier from wild animal to human. SARS develops within days after infection by a positive-stranded RNA coronavirus (SARS-CoV), which has proven to be highly virulent and cytotoxic. The engine driving the replication of the SARS-CoV is a large complex of replicase proteins that involves up to 15 individual proteins essential for the viral life cycle. Many of these proteins have no assigned function or sequence homology to other known proteins. Currently, the only experimentally determined structure of a SARS-CoV protein is that of the chymotrypsin-like protease. The overall goal of this proposal is to determine the structures of individual proteins that form the SARS-CoV replicase complex and to use the structures as a foundation to understand their biological functions. In addition, the structures of homologous replicase proteins from the mouse hepatitis virus (MHV), a closely related virus that does not infect humans, will be determined and compared to those of the SARS-CoV. The first specific aim of this proposal is to clone, express and purify replicase proteins of the SARS-CoV and MHV for crystallographic analysis. Currently, we have clones for 15 of 18 target proteins from SARS-CoV and MHV, of which 13 have been expressed solubly and 6 have been affinity purified. The second specific aim of this proposal is to crystallize these replicase proteins and determine their structures using the technique of X-ray diffraction. High-throughput robotic crystallization screens have been performed on 6 target proteins. Crystals of the p12 domain of the SARS-CoV were obtained, diffracted to 6 A resolution, and cell constants determined. The information generated by the comparison of SARS-CoV and MHV will be instrumental in understanding biological differences such as species specificity (human vs. animal) and virulence (cytotoxicity and infectivity) in these viruses and may be applicable to other virus families. In collaboration with Dr. Mark Denison of Vanderbilt University, we will generate structure based hypotheses that will be tested in SARS-CoV and MHV reverse genetics systems developed in his laboratory.